caa a22 4500 445882964 CHVBK 20180317145550.0 cr unu---uuuuu 170323e20110601xx s 000 0 eng 10.1007/s10157-010-0399-4 doi (NATIONALLICENCE)springer-10.1007/s10157-010-0399-4 Antigen-binding abilities of anti-nephrin antibody are prescribed by signal sequence of expression vector in genetic immunization [Elektronische Daten] [Shokichi Naito, Kouju Kamata, Togo Aoyama, Nozomu Yamanaka, Tomoko Okamoto, Hiromi Tazaki] Background: Nephrin is an essential protein for maintaining the normal structure of podocyte foot processes and the glomerular filtration barrier. To analyze the mechanism of proteinuria and nephrotic syndrome, we previously reported on a new method of producing polyclonal anti-nephrin antibody by genetic immunization. In the present paper, we investigate the effect of signal peptide sequence cDNA on the characteristics of polyclonal anti-nephrin antibodies induced by genetic immunization. Methods: Five fragments of nephrin cDNA with or without signal peptide sequence were inserted into the pTARGET™ vector. Rats were immunized with these vectors by the gene gun method. Sera obtained from rats at 14weeks following immunization were analyzed by Western blotting, immunoprecipitation, flow cytometry and immunohistochemistry. Results: Four different antibodies induced by cDNA encoding nephrin protein fragments without signal peptide showed antigen site-specific binding to fragmented glycosylation-disturbed nephrin proteins. These antibodies also reacted to a glycosylation-disturbed full-length nephrin protein (inhibited by tunicamycin), but did not react to either a native nephrin protein or a fully glycosylated conformational nephrin protein. Four different antibodies induced by cDNA encoding nephrin fragments with signal peptide showed an antigen site-specific binding to glycosylation-disturbed nephrin protein fragments. In addition, these antibodies reacted to both a native nephrin protein and a full-length glycosylated conformational nephrin protein. Conclusions: The absence of signal peptide sequence cDNA in the expression vector produced antibodies specific for glycosylation-disturbed proteins, while its presence produced antibodies that bound to native or fully glycosylated conformational protein. Japanese Society of Nephrology, 2011 Antibody specificity nationallicence Genetic immunization nationallicence Nephrin nationallicence N-glycosylation nationallicence Signal peptide nationallicence Naito Shokichi Department of Internal Medicine, Kitasato University Graduate School of Medical Sciences, 252-0374, Sagamihara, Kanagawa, Japan aut Kamata Kouju Department of Internal Medicine, Kitasato University Graduate School of Medical Sciences, 252-0374, Sagamihara, Kanagawa, Japan aut Aoyama Togo Department of Internal Medicine, Kitasato University Graduate School of Medical Sciences, 252-0374, Sagamihara, Kanagawa, Japan aut Yamanaka Nozomu Department of Surgery, National Defense Medical Collage, Tokorozawa, Saitama, Japan aut Okamoto Tomoko Department of Internal Medicine, Kitasato University Graduate School of Medical Sciences, 252-0374, Sagamihara, Kanagawa, Japan aut Tazaki Hiromi Department of Internal Medicine, Kitasato University Graduate School of Medical Sciences, 252-0374, Sagamihara, Kanagawa, Japan aut Clinical and Experimental Nephrology Springer Japan 15/3(2011-06-01), 339-345 1342-1751 15:3<339 2011 15 10157 https://doi.org/10.1007/s10157-010-0399-4 text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1007/s10157-010-0399-4 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Naito Shokichi Department of Internal Medicine, Kitasato University Graduate School of Medical Sciences, 252-0374, Sagamihara, Kanagawa, Japan aut NATIONALLICENCE 700 1- Kamata Kouju Department of Internal Medicine, Kitasato University Graduate School of Medical Sciences, 252-0374, Sagamihara, Kanagawa, Japan aut NATIONALLICENCE 700 1- Aoyama Togo Department of Internal Medicine, Kitasato University Graduate School of Medical Sciences, 252-0374, Sagamihara, Kanagawa, Japan aut NATIONALLICENCE 700 1- Yamanaka Nozomu Department of Surgery, National Defense Medical Collage, Tokorozawa, Saitama, Japan aut NATIONALLICENCE 700 1- Okamoto Tomoko Department of Internal Medicine, Kitasato University Graduate School of Medical Sciences, 252-0374, Sagamihara, Kanagawa, Japan aut NATIONALLICENCE 700 1- Tazaki Hiromi Department of Internal Medicine, Kitasato University Graduate School of Medical Sciences, 252-0374, Sagamihara, Kanagawa, Japan aut NATIONALLICENCE 773 0- Clinical and Experimental Nephrology Springer Japan 15/3(2011-06-01), 339-345 1342-1751 15:3<339 2011 15 10157 Metadata rights reserved Springer special CC-BY-NC licence nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-springer